Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. Glutamatergic signaling between neurons is critically regulated by neighboring astrocytes, which surround most synaptic junctions and express specific glutamate transporters that control glutamate concentrations by removing glutamate from the synaptic cleft. Astrocytes also express glutamate receptors, specifically the metabotropic glutamate receptor subtypes mGluR3 and mGluR5, which are activated by glutamate and known to regulate glutamate transporter activity. Whereas much is known about the regulation and sorting of glutamate receptors in neurons, very little is known about the potential for specialized regulation of glutamate receptors and transporters in astrocytes. The mGluR subtypes 3 and 5, as well as the astrocytic glutamate transporter EAAT1, all possess large intracellular carboxyl-termini (CT) that play key roles in the control of their activity. Since these CTs terminate in consensus motifs for potential association with a class of conserved protein-protein interaction domains known as PDZ domains, we screened a custom-made PDZ domain proteomic array and found that the CTs of mGluR3, mGluR5 and EAAT1 all exhibit robust and specific interactions with the PDZ domains of the multifunctional scaffold protein NHERF-2. These interactions were confirmed in a cellular context, and immunohistochemical studies revealed that NHERF-2 is abundantly expressed in astrocytes in the brain. We hypothesize that NHERF-2 is a central regulator of the activity and localization of mGluRs and EAAT1 in astrocytes, and may facilitate mutual regulation between mGluRs and glutamate transporters. We will test this idea by examining NHERF-2 regulation of mGluR3, mGluR5 and EAAT1 functional activity and cross-talk, as well as by exploring the possibility that NHERF-2 may control mGluR and EAAT1 localization in vivo by performing immunohistochemical analyses on brain tissue from wild-type versus NHERF-2 knockout mice. These studies are of significant clinical importance because metabotropic glutamate receptors and glutamate transporters are considered to be excellent potential therapeutic targets in the treatment of stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, schizophrenia, and other brain disorders. PUBLIC HEALTH RELEVANCE Astrocytes and neurons are intermingled throughout the brain, and astrocytes possess receptors that can sense neuronal activity as well as transporters that can regulate the levels of synaptic neurotransmitters. Astrocytic receptors and transporters are often clustered near synapses and known to be highly regulated, but the mechanisms underlying this targeting and regulation are largely unknown. The work described in this application will explore the mechanisms by which astrocytic receptors and transporters, specifically those that respond to the neurotransmitter glutamate, are targeted and regulated. These studies are of significant clinical importance because astrocytic glutamate receptors and transporters are considered to be excellent potential therapeutic targets in the treatment of stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, schizophrenia, and other brain disorders.